establishing a mouse model of small fiber neuropathy using resiniferatoxin

Establishing a Mouse Model of Small Fiber Neuropathy Using Resiniferatoxin

To begin, put on personal protective equipment and take proper precautions while making the resiniferatoxin or RTX stock solution. Dilute one aliquot of 600 microliters in saline for the experiment, and divide the rest into 12-microliter aliquots for storage. For test mice, use eight-week-old adult male ICR mice, weighing between 35 and 40 grams. Administer a single dose of RTX solution intraperitoneally with a microinjection syringe. Next, inject a second group of mice with an equal volume of vehicle to serve as a control. After making all of the injections, house the mice in their home cages, and be certain that they have ad-lib access to food and water.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Establishment of Resiniferatoxin (RTX) Neuropathy
CAUTION:&#160;RTX is neurotoxic and hazardous. On contact, it acts as an irritant to the eyes, mucous membranes, and upper respiratory tract. Avoid inhalation and wear lab eyeglasses and coats during RTX preparation. Rinse with plenty of water in case of skin contact or after handling.
<ol>
	<li>Add 1 mg of RTX powder to a 200 &#181;L mixture of equal volume of Tween 80 and absolute ethanol (100 &#181;L for each solvent).</li>
	<li>Aliquot the RTX solution (12 &#181;L/vial) and store it at -20 &#176;C for up to 3 months. This constitutes the RTX stock; discard the remaining RTX solution at expiration.</li>
	<li>Dilute the RTX stock with normal saline to a final volume of 600 &#181;L. The final concentration of the RTX solution should be 0.01%, which equals 1 &#181;g RTX in 10 &#181;L vehicle solution.</li>
	<li>Use 8-week-old adult male ICR mice (35-40 g) as experimental animals and administer a single dose of RTX solution (dose: 200, 50, and 10 &#181;g/kg, respectively) intraperitoneally (i.p.) with a microinjection syringe to the mice.&#160;Mice were anesthetized by inhaler with 5 % isoflurane for deep anesthesia. If mice showed withdrawal action of extremities during RTX injection, mice should take longer inhalation of anesthesia.&#160;&#160; 
	Example: If the mouse weighs 40 g, then it will receive 20 &#181;L of the RTX solution, representing the dose of 50 &#181;g/kg.</li>
	<li>Give one group of mice an equal volume of vehicle (10% Tween 80 and 10% absolute ethanol in saline solution) as a control.</li>
	<li>After RTX injection, return the mice to a plastic cage on a 12-h light/12-h dark cycle and provide food and water&#160;ad libitum.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resiniferatoxin</td>
			<td>Sigma</td>
			<td>R8756</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma</td>
			<td>P1754</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Lee, Y. et al., <a href="https://app.jove.com/v/56651">Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1.</a>&#160;J. Vis. Exp.&#160;(2018).
This video demonstrates a method for establishing a small fiber neuropathy model in mice using the TRPV1 agonist resiniferatoxin (RTX). It outlines the steps involved in administering RTX to mice and explains how RTX induces neuronal death in small-diameter sensory neurons, thereby establishing the neuropathy model.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a solution of resiniferatoxin (RTX), a high-affinity agonist of the TRPV1 receptor&#8212;pain-sensitive ion channels expressed on sensory neurons.
Take an anesthetized adult mouse and administer RTX intraperitoneally.
The drug enters systemic circulation and reaches the dorsal root ganglia (DRG), a cluster of sensory neuron cell bodies located near the spinal cord.
RTX crosses the blood-nerve barrier and interacts with small-diameter sensory neurons in the DRG that abundantly express TRPV1.
RTX binding to TRPV1 activates the receptors, inducing a massive calcium ion influx into the neurons.
Intracellular calcium activates enzymes that degrade cell membrane phospholipids, cellular proteins, and DNA, ultimately leading to neuronal death.
Subsequently, the intraepidermal nerve fibers (IENFs), the peripheral terminals of small-diameter sensory neurons that innervate the skin, begin to degenerate.
This degeneration of IENFs results in the loss of sensory input from the skin, thereby establishing a small fiber neuropathy model.

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Establishing a Mouse Model of Small Fiber Neuropathy Using Resiniferatoxin

Transcription

Establishing a Mouse Model of Small Fiber Neuropathy Using Resiniferatoxin